The Genetic Variability of UCP4 Affects the Individual Susceptibility to Late-Onset Alzheimer’s Disease and Modifies the Disease’s Risk in APOE- ɛ 4 Carriers

Abstract

Uncoupling proteins (UCPs) are a group of five mitochondrial inner membrane transporters with a tissue specific expression that uncouple biofuel oxidation from ATP synthesis and function as regulators of energy homeostasis and antioxidants. Previous data suggested that neuronal UCPs (UCP2, UCP4, and UCP5) can directly influence synaptic plasticity, neurotransmission, and neurodegenerative processes, and have a crucial role in the function and protection of the central nervous system. In fact, it has been observed that the expression of neuronal UCPs significantly decreases in Alzheimer’s disease (AD) patients. Here we analyzed the variability of UCP2, -3, -4, and 5 genes in sporadic and familial cases (n = 465) of late-onset AD (LOAD) with respect to healthy controls (n = 442). We showed that a genetic variant in the human UCP4, rs9472817, not only significantly affects the individual susceptibility to LOAD, but also modulates the effect of APOE-ɛ4 on AD risk. In fact, rs9472817-C allele was significantly more frequent in both groups of patients with respect to the control group (p = 6.934^*10^–4 for familial and p = 1.033^*10^–3 for sporadic cases). In addition, gene-gene interaction analysis revealed that the effect of APOE-ɛ4 allele on LOAD risk was doubled in homozygote CC subjects; conversely, the risk conferred by the APOE-ɛ4 allele was annulled in subjects with two copies of the G allele. Our findings are further evidence that the efficiency in mitochondrial energy metabolism and oxidative stress are important factors in AD pathogenesis.

Keywords

Alzheimer’s disease APOE genetic risk mitochondria neurodegeneration UCP4 uncoupling proteins

INTRODUCTION

Alzheimer’s disease (AD) is the main cause of dementia among elderly population, affecting millions of people worldwide. AD is classified as early-onset AD or late-onset (LOAD) depending if dementia appears before or after the age of 65. LOAD, the most common type, is usually sporadic. However, genetics of late-onset forms is complex, with susceptibility likely determined by many common but weakly penetrant genetic variants interacting with environmental and epigenetic factors. Increasing age and the ɛ4 allele of the apolipoprotein E (APOE) are the strongest and most consistently replicated risk factors for LOAD development [1].

A growing body of evidence indicates that mitochondrial dysfunction and oxidative stress occur before the clinical manifestations become evident, thus playing an important role in the early pathogenetic phases of the disease [2 –4]. In fact, several mitochondrial defects, including impaired bioenergetics, increased oxidative stress, altered calcium buffering capacity, and abnormal mitochondrial dynamics have been described in patients and animal models for the disease [5 –11]. Moreover, mitochondria intervene in the mechanisms by which intracellular amyloid-β triggers synaptic failure and neurodegeneration interacting with different mitochondrial components and progressively accumulating within mitochondria (see the reviews [12, 13] and references therein). Also hyperphosphorylated tau leads to impaired mitochondrial function suggesting that tau and amyloid-β synergistically impair mitochondria, accelerating the neurodegenerative process [14 –16].

The involvement of mitochondrial function in AD pathogenesis is further supported by association studies, which suggest that variants of genes coding for mitochondrial proteins are likely to influence the onset and the outcome of the disease. For example, variants of TOMM40 [17], TFAM [18, 19], COX10, and COX15 [20] genes, as well as variations in mitochondrial DNA [21] appear to be linked to variability of the individual risk to develop AD.

In the last years, the uncoupling activity of mitochondria, namely the uncoupling of oxidative phosphorylation from ATP production, is emerging as one of the key players in the regulation of mitochondrial energy metabolism and an important determinant, through its dysfunctions, of metabolic disorders. The uncoupling process is mediated mainly by uncoupling proteins (UCPs), a subfamily of mitochondrial anion-carriers localized in the inner mitochondrial membrane, which transport protons directly from the intermembrane space to the matrix, thereby dissipating the electrochemical proton gradient generated by respiration as heat [22]. Five UCPs (named UCP1 to 5) are known in humans with similarities in their structure, but tissue-specific expression. Neurons express at least three UCPs including the widely expressed UCP2 and the neuron-specific UCP4 and UCP5 [23]. There are strong indications that these proteins through the mediation of a “mild uncoupling” may reduce the formation of reactive oxygen species (ROS) without significantly affecting the ATP synthesis, thereby acting as modulators of oxidative metabolism [24, 25]. The fact that neurons are highly dependent on oxidative phosphorylation and particularly sensitive to energy deficits and oxidative stress has suggested that many of the mechanisms leading to mitochondrial-induced neuronal dysfunction might be regulated by the activity of these neuronal UCPs [23]. This has gathered considerable support by experimental evidence showing, for instance, that the thermogenic activity of UCP2 in neurons modulates neural transmission in synapses [26], and that its expression induces the decrease of ROS generation in brain of UCP2-knockout mice [27]. In vitro studies have shown that the overexpression of UCP4 increased cell survival and this was concomitant to reduced oxidative stress and increased ATP synthesis (it is controversial whether the effect on ATP synthesis occurs because of an adaptive shift in energy metabolism, from mitochondrial respiration to glycolysis, or because of an increased Complex II activity) [28 –31]. UCP4 can also regulate calcium homeostasis, and its knockdown in primary hippocampal neurons results in calcium overload and cell death [28, 32]. Interestingly a genetic variant of UCP4 has been found associated to leukoaraiosis [33]. More directly, the expression of UCP2, 4, and 5 has been reported to significantly decrease in AD patients, and this was associated to an increased oxidative stress and mitochondrial dysfunction [34].

Here we report an investigation on the contribution of genetic variability of neuronal UCP genes to the risk of LOAD. Since UCP2 and UCP3 genes are located within 8 kb of each other, we also included UCP3 in our analysis. Sixteen SNPs were analyzed in a sample of 465 LOAD cases and 442 ethnicity-matched cognitively healthy controls.

MATERIALS AND METHODS

Sample

465 unrelated patients diagnosed as affected by LOAD (162 men and 303 women) recruited at the Regional Neurogenetics Centre (Calabria, Southern Italy) were analyzed in the present study.

According to familiarity (at least one first-degree relative with AD), the cases were classified as affected by familial (n = 264 subjects, by including in the study only one case per family) or sporadic LOAD (N = 201 subjects).

All patients underwent a detailed clinical assessment involving medical history, physical and routine laboratory examinations, including serum folate, vitamin B12, thyroid function, and syphilis serology. Activities of daily living and instrumental activities of daily living were assessed in all patients. Cognitive status was investigated through the Mini-Mental State Examination (MMSE) [35]. MMSE scores were adjusted for age and educational level according to procedure reported in a study by Magni et al. [36]. Clinical diagnosis for AD was performed through criteria of the National Institute on aging and the Alzheimer’s Association workgroup [37].

Brain imaging (CT-MRI) was performed. McKeith criteria [38], clinical and neuropathological criteria for frontotemporal dementia [39], and NINDS-AIREN criteria [40] were used to differentiate Lewy body dementia, frontotemporal dementia, and vascular dementia from AD.

The control group represented by 442 unrelated subjects (225 men and 217 women) was recruited in the same population of LOAD patients, paying attention to match cases and controls for age, ethnicity, and origin in the area, as ascertained by genealogical analyses carried out on three generations.

An informed written consent was signed by all subjects or their legal representative. This study was performed according to the Declaration of Helsinki with appropriate ethics committee approval.

SNP selection

SNPs were selected from literature data, and using information from public databases (http://www.ncbi.nlm.nih.gov/; http://www.hapmap.org/). The selection was based on the following criteria: Minor allele frequency (MAF) >5% in Caucasians, putative functional significance (non-synonymous SNPs, SNPs located in the 5’ - and 3’ – UTR regions), SNPs previously investigated in association studies. For each gene the selected SNPs are reported in Supplementary Table 1.

Genotyping using the Sequenom iPLEX ^TM assay

Multiplex SNP genotyping was performed using PCR followed by primer extension and MALDI-TOF mass spectrometry using iPLEX Gold technology from Sequenom (Sequenom Inc, San Diego, USA). Sequenom MassARRAY Assay Designer software (version 3) was used to design primers for PCR and single base extension. Standard procedures were used to amplify PCR products, and unincorporated nucleotides were deactivated with the shrimp alkaline phosphatase (SAP). A primer extension reaction was subsequently implemented using the mass extension primer and the terminator. The primer extension products were then desalted on resin, and spotted onto the 384-element SpectroCHIP (Sequenom) for MALDI-TOF analysis using SpectroACQUIRE v3.3.1.3 (Sequenom). Spectra were analyzed using MassARRAY Typer v3.4 Software (Sequenom).

Approximately 10% of the samples were analyzed in duplicate, and the concordance rate of the genotypes was higher than 99%.

APOE genotyping (alleles ɛ2, ɛ3, and ɛ4) was carried out according to the protocol described in a study carried out by Carrieri et al. [41].

Genetic and statistical analyses

For each analyzed polymorphism, allele and genotype frequencies were estimated by gene counting from the observed genotypes. Hardy– Weinberg equilibrium was tested by Fisher’s exact test.

The association between the analyzed genetic variants and the disease phenotype was assessed by fitting for each SNP the following logistic regression model: $ln \frac{p}{1 - p} = β_{0} + β_{1} sex + β_{2} SNP (model 1)$ where p represents the probability of belonging to the AD group; β_0, β_1, and β₂ are parameters to be estimated; Sex is the sex of the subject (1 for males and 2 for females); and SNP represents the analyzed polymorphism coded in an additive fashion (number of copies of the minor allele). In such model, in order to test if β₂ was significantly different from 0 the Wald’s test was used.

To capture the sex-dependent effects of the analyzed genetic variants, model 1 can be reformulated as follows:

$\begin{matrix} ln \frac{p}{1 - p} = β_{0} + β_{1} sex + β_{2} SNP + β_{3} sex \\ \times SNP (model 2) \end{matrix}$ where β₃ is the coefficient of the interaction effect; it indicates how the effect of the SNP on the phenotype of interest changes between males and females.

To capture gene-gene interaction, model 1 can be reformulated as follows:

$\begin{matrix} ln \frac{p}{1 - p} = β_{0} + β_{1} sex + β_{2} {SNP}_{1} + β_{3} {SNP}_{2} \\ + β_{4} {SNP}_{1} \times {SNP}_{2} (model 3) \end{matrix}$ where β₄ is the coefficient of the interaction effect; it indicates how the effect of the SNP₁ on the phenotype of interest changes with respect the strata defined by SNP₂ genotypes.

In both these cases, to assess if β₃ in model 2 and β₄ in model 3 were significantly different from 0 the Wald’s test was used. Adjustment for multiple comparisons has been carried out using the Bonferroni procedure

In order to visually synthesize the association results obtained in the present study the Synthesis-View software was used [42].

Single-locus analysis was carried out using Plink v1.07 [43]. The analysis and visualization of LD between the analyzed loci was performed using Haploview [44].

RESULTS

Table 1 shows the demographic and clinical characteristics of the population sample included in this study. The genotype distributions at all SNPs typed were in Hardy-Weinberg equilibrium in controls (p > 0.05), with the exception of rs15763 (UCP2) and rs2235800 (UCP5) which were excluded from further analysis. Table 2 and Figure 1 show the results of the logistic regression analysis. Two of the 16 SNPs, rs2734827-CT in UCP3, and rs9472817-CG in UCP4, showed nominally statistically significant associations (p≤0.05) with LOAD risk in both familial and sporadic cases, while rs3007756-GA in UCP5 was nominally associated with the disease risk only in familial cases. After Bonferroni adjustment for multiple testing, the association with the disease was still significant for UCP4-rs9472817 (p < 0.05/16).

Regarding the SNP rs9472817, the minor G allele appeared to act as a protective factor for AD in both familial (OR = 0.668, CI: 0.529–0.843; p = 6.934^*10^–4) and sporadic (OR = 0.651, CI: 0.504–0.841; p = 1.033^*10^–3) cases. Conversely, the C allele appeared to act as an additive risk factor. In fact, in familial LOAD patients, with respect to subjects with the GG genotype (no risk allele), the estimated risk of developing the disease for subjects with the CG genotype (1 copy of the risk allele C) was increased of about 1.50-fold, while subjects with two copies of the some allele (homozygous CC) had an almost 2.24– fold increased risk This gene-dosage dependent effect was evident also in sporadic LOAD patients, where subjects with the CG and CC genotypes showed, respectively, a 1.53-fold and 2.36-fold risk of developing the disease when compared to subjects with the GG genotype. Figure 2 clearly shows the additive effect played by the C allele on the AD risk in familial (A) and sporadic (B) cases. The results obtained were not influenced by gender, age at disease-onset, or by the level of cognitive impairment (measured by MMSE).

The lower panel of Fig. 1 shows the pattern of LD between SNPs in each gene region. We found no LD between rs9472817 and the other tested SNPs in the UCP4 gene; consequently, no haplotype analysis was performed.

APOE-ɛ4 and UCP4-rs9472817 interaction

As reported in Table 3, and well documented in literature data, we found an increased risk of AD in APOE-ɛ4 allele carriers in both familial (OR = 9.423, p = 1.61^*10^–23) and sporadic cases (OR = 6.339; p-value = 1.86^*10^–14). Interestingly, when this association was analyzed in different strata defined according to UCP4-rs9472817 genotypes (Table 3 and Supplementary Table 1), we observed an important increase in the OR associated with the presence of the C allele in both groups of patients. Specifically, as it regards the subgroup of familial LOAD patients, the highest OR was found in rs9472817-CC homozygous (OR = 19.857, p = 2.83^*10^–9), an intermediate OR in heterozygous CG (OR = 12.332, p = 1.02^*10^–13), while the lowest OR was found in rs9472817-GG homozygous subjects (OR = 2.525, p = 0.060). A similar trend was observed for sporadic cases. In facts, the estimated ORs were 12.068 (p = 2.00^*10^–5), 7.182 (p = 3.53^*10^–8), and 2.577 (p = 0.086), for the rs9472817-CC, CG and GG genotypes respectively. All together, these results indicate that the C allele acts in an additive manner to increase the AD risk due to the presence of the APOE-ɛ4 allele. Moreover, it is worth to notice that the presence of two copies of the G allele nullified the risk conferred by APOE-ɛ4 allele (p > 0.05 in both group of patients). These findings prompted us to explore the potential genetic interaction between APOE-ɛ4 status and rs9472817 genotypes. A logistic regression model including the interaction term “rs9472817 C-carriers^* APOE-ɛ4 carriers” provided estimated p-values for the interaction coefficients (see model 3 in Materials and Methods) of 0.006 and 0.054, respectively for familial and sporadic cases, that confirmed the above results.

DISCUSSION

The idea that mitochondria play an important role in LOAD stems from the fact that these organelles are key regulators of cell viability and perform functions (i.e., control of energy, ROS, and calcium homeostasis) that are essential for the brain [45, 46].

Our motivating hypothesis was that the activity of the neuronal mitochondrial UCPs could be effective in protecting neurons from degeneration by limiting the production of ROS and/or by regulating energy utilization, and, consequently, genetic variations in these UCPs loci could account for a proportion of the LOAD genetic risk. In examining this hypothesis, we detected an allele dose-dependent effect on LOAD susceptibility at rs9472817-CG of UCP4, in both familial (p = 6.934^*10^–4) and sporadic (p = 1.033^*10^–3) cases, where the presence of the C allele behaves as a risk factor while G is protective.

This association, which appears to be in contrast with a previous study where the G allele was found detrimental for longevity [47], may be related to the reported role of UCP4 in regulating mitochondria activity and in helping neurons to cope with conditions of metabolic and oxidative stress [28 –31]. However, the exact role of this gene in the context of AD is not currently known, although some studies implicate UCP4 in the disease pathophysiology. For instance, the up-regulation of UCP4 protein levels as well as the UCP4-dependent upregulation of mitochondrial free calcium in response to exposure to the superoxide treatment were found to be diminished in cells overexpressing AβPP or AβPP mutant [48]. Moreover, the expression of UCP4 was found significantly decreased in the brain of AD patients [34]. Our study, for the first time, to the best of our knowledge, revealed that the variability of UCP4 contributes to the risk of developing AD, providing further support for a role of this gene in the disease susceptibility.

At present, the biological significance of the rs9472817 polymorphism is unknown. Considering its intronic location, it is likely that it is not the causal variant but rather tags another functional SNP within the UCP4 gene or in another nearby gene. We used linkage disequilibrium (LD) information from the 1000 Genomes Project to search SNPs in LD (cutoff of r² = 0.8) with rs9472817. We found that rs9472817 is in LD with 13 currently known SNPs, spanning a region of about 71.5Kb encompassing the genes CYP39A1 and PLA2G7. Therefore, we cannot exclude that one of these common SNPs might be the actual causal variant. Alternatively, the observed association could be related to the so-called phenomenon of synthetic association, which raises the possibility that common genetic variants may be coupled with functional rare variants tagged by the common ones [49]. Accordingly, the association with the common SNP rs9472817 could reflect the net phenotypic effect of low-frequency causal variants distributed broadly within the gene. This might lead, in some cases, to contrasting results due to the occurrence of rare mutations with different effects in different population groups, and this might explain the contrast of the present results with those reported in the study conducted by Rose and associates [47]. The complete sequencing of numerous mtDNA molecules showed, for instance, that mutations in subunits of the complex I have a beneficial effect on longevity, while the simultaneous presence of mutations in complex I and III or V seems to be detrimental [50].

Regardless, considering that almost all the LOAD susceptibility risk alleles so far identified typically have ORs of 1.5 or less, we believe the results of our study may provide an important contribution to the understanding of the genetic risk in AD, shedding light to the current missing hereditability for AD.

Epistatic interactions may account for much of the unexplained variance in AD status [51 –53], so we investigated the effect of interaction between variants at the UCP4 and APOE loci on LOAD risk. Besides confirming the risk association between APOE-ɛ4 and LOAD, our data provide evidence that UCP4 and APOE interact to modify the risk of developing the disease. In APOE-ɛ4 carriers that also carried two copies of rs9472817-C allele, the risk of LOAD was two-fold the risk of those that carried APOE-ɛ4 allele alone (OR = 19.857 versus OR = 9.423, in familial cases; OR = 12.068 versus OR = 6.339, in sporadic cases); while, the risk conferred by APOE-ɛ4 allele was nullified by the presence of two copies of the G allele (OR ∼ 2.5 in both subgroup of patients; p > 0.05).

However, it should be specified that the p value for the gene-gene interaction coefficient was statistically significant in familial cases (p = 0.006) and marginally significant in sporadic ones (p = 0.054). We believe that this could mostly be due to the lower risk conferred by APOE-ɛ4 in sporadic rather than in familial cases (ORs = 6.339 versus 9.423).

There are several reports of variants interacting with APOE genotypes; however, their effects in modifying the risk due to APOE-ɛ4 allele is quite modest [54 –59]. Our results support the role of UCP4 rs9472817 as one of the most relevant genetic modifiers of the risk conferred by APOE-ɛ4 reported to date. We are aware that evidence of a statistical interaction does not necessarily imply an underlying biological interaction. However, there are various molecular pathways through which these two risk factors may jointly affect the disease risk. For instance, the ability of UCP4 to modulate ROS production and glucose metabolism in the brain could be the molecular links to explain the interaction with APOE. This hypothesis is supported by the fact that APOE has been shown to have an isoform-specific antioxidant activity, with APOE-ɛ4 showing the lowest antioxidant potential [60, 61], and by findings consistent with APOE-ɛ4 effects on cerebral glucose metabolism, with APOE-ɛ4 carriers having reduced glucose metabolic rates compared to non-carriers [62, 63]. On the other hand, also the ability of UCP4 to regulate Ca^2 + homeostasis could explain the interaction with APOE, given that: (i) aberrant calcium dysregulation has been consistently implicated in AD [64]; (ii) the neurotoxic effects of APOE-ɛ4 are mediated by an alteration in calcium homeostasis [65]; (iii) in old age high serum calcium levels are associated with worse cognitive function, especially in APOE-ɛ4 allele carriers [66].

In conclusion, we found that the variability of UCP4 does affect the individual susceptibility to LOAD. In particular, the rs9472817-C allele increases the susceptibility to LOAD and increases the penetrance of APOE-ɛ4 allele. At this stage we cannot explain in detail the molecular mechanism underlying these findings, and additional studies will be needed to clarify this point. In any case, our study does confirm the importance of mitochondrial functionality in the pathogenesis of AD.

Footnotes

ACKNOWLEDGMENTS

We are grateful to patients and families for the interest and generous participation in our research effort and the Associazione per la Ricerca Neurogenetica-ONLUS Lamezia Terme for invaluable help in assisting persons and families.

This work was partially supported by the European Union’s Seventh Framework Programme (FP7/2007-2011) [grant number 259679], by funds from Programma Operativo Nazionale [01_00937] - MIUR“Modelli sperimentali biotecnologici integrati per lo sviluppo e la selezione di molecole di interesse per la salute dell’uomo”, and by the Italian Health Ministry (DGRST number 4/2760-P/I.9 ab, 2007; RFPS-2006-7-334858, 2006).

Authors’ disclosures available online ().

Appendix

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The Genetic Variability of UCP4 Affects the Individual Susceptibility to Late-Onset Alzheimer’s Disease and Modifies the Disease’s Risk in APOE- ɛ 4 Carriers

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Sample

SNP selection

Genotyping using the Sequenom iPLEX TM assay

Genetic and statistical analyses

RESULTS

APOE-ɛ4 and UCP4-rs9472817 interaction

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

Appendix

References

Genotyping using the Sequenom iPLEX ^TM assay